Tublin, the principal component of intracellular microtubules, interacts with associated proteins, nucleotides and metal ions that regulate the assembly and disassembly of microtubules during various stages of cell growth and differentiation. An understanding of these control processes will require information on the three-dimensional structure of tubulin at resolutions approaching the atomic. The primary project proposed concerns the study of extended two-dimensional lattices of tubulin molecules by advanced electron microscopy to obtain such structural information. The lattices are formed in the presence of zinc which converts tubulin assemblies from helical to flat arrays. The electron microscopy involves low-dosage methods on unstained samples, with structural data extracted from micrographs by image reconstruction in conjunction with tilting of sample grids. Studies already completed in this laboratory extend the limit of resolution on negatively stained samples to about 15 A and demonstrate the feasibility of measurements on unstained samples. Two secondary projects are also proposed, concerning the kinetic mechanism of microtubule assembly and the energetics of the interactions between the alpha and beta polypeptide chains of 6S tubulin. For the kinetic studies, we will monitor the assembly of microtubules by turbidity as a function of concentration, nucleotides, metal ions and associated proteins. From the shape of the progress curves and the structures of intermediates determined by electron microscopy, conclusions will be drawn about the mechanism of assembly. Energetics of the interactions within 6S tubulin will be studied by ultracentrifugaton with sedimentation monitored by fluorescence using an optical system constructed in this laboratory. Initial experiments at the low concentrations that can be achieved with this optical system reveal a dissociation of 6S tubulin into individual alpha and beta subunits. Observations on the dissociation constant under various conditions can thus be used to characterize conformational states.